[go: up one dir, main page]

EP0788605B1 - Detection d'anticorps contre des antigenes bacteriens par polarisation de fluorescence - Google Patents

Detection d'anticorps contre des antigenes bacteriens par polarisation de fluorescence Download PDF

Info

Publication number
EP0788605B1
EP0788605B1 EP96933797A EP96933797A EP0788605B1 EP 0788605 B1 EP0788605 B1 EP 0788605B1 EP 96933797 A EP96933797 A EP 96933797A EP 96933797 A EP96933797 A EP 96933797A EP 0788605 B1 EP0788605 B1 EP 0788605B1
Authority
EP
European Patent Office
Prior art keywords
antigen
fluorophore
antigens
bacterial
conjugated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP96933797A
Other languages
German (de)
English (en)
Other versions
EP0788605A1 (fr
Inventor
Michael E. Jolley
Klaus H. Nielsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diachemix LLC
Original Assignee
Diachemix LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Diachemix LLC filed Critical Diachemix LLC
Publication of EP0788605A1 publication Critical patent/EP0788605A1/fr
Application granted granted Critical
Publication of EP0788605B1 publication Critical patent/EP0788605B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • a foreign antigenic substance in the body typically gives rise to an immune response.
  • Antibodies specific for the substance are secreted into the blood stream. Diagnostic tests revealing exposure of an animal to an infectious agent rely upon in vitro detection of antibodies specific for antigens expressed by such an infectious agent.
  • antibodies contained in a serum sample are brought into contact with an antigen preparation.
  • the resulting immune complex is detected in a variety of assay formats including enzyme-linked immunosorbant assay (ELISA), agglutination tests, complement fixation assay, etc.
  • the assay formats involve adsorption of antigens into a solid phase of such as a plastic bead or particle, a plastic well, or the surface of a plastic or glass tube.
  • the serum containing antigen-specific antibodies are contacted within antigen coated onto solid phase, and washed to remove unreacted serum components.
  • the solid phase-bound antibodies are then detected, for example, by reacting with a species-specific antibody bearing a reporter molecule, washing to remove unreacted reagent, and finally adding a reagent to activate the reporter.
  • bovine brucellosis This disease is caused by Brucella abortus , a Gram negative bacterium inhabiting the genital tract of cattle. Infected heifers are highly susceptible to abortion, and the economic loss to the cattle industry has been prodigious. Through concerted international efforts to screen for infected animals, and disposal of test positive animals at slaughter, the disease is essentially eradicated in Canada, and the incidence in the U.S. has diminished to less than 150 infected herds. However, Mexico is a persistent reservoir of infection, and movement of cattle in commerce to the U.S. poses a continual risk of new disease. Also, wild animals such as deer and elk are a reservoir of potential new disease in domestic livestock.
  • Brucellosis is but one example of veterinary disease for which a need exists for a sensitive yet highly specific assay approaching 100 percent.
  • Other diseases include tuberculosis (M. bovis ), bovine leukosis, leptospirosis, foot and mouth disease (Africa and South America), and salmonellosis.
  • the fluorescent conjugate is designed to provide insight into specific ligand-amino acid interactions in oligosaccharide-antibody complexes and to enable determination of the thermodynamics of binding by the technique of fluorescence quenching.
  • EP-A-0 265 127 discloses an assay in which a lipopolysaccharide component of an endotoxin binds to a cyclic peptide.
  • a label is applied to one of the binding partners.
  • the labels may be enzyme labels, radiolabels, or fluorescent labels.
  • WO-A-96 22534 discloses an assay in which an endotoxin, labeled with ruthenium (II) tris-bipyridine NHS ester, binds with polymyxins. Electrochemiluminscence is used for detection.
  • a fluorophore-conjugated antigen dissolved freely in solution reacts with antibodies contained in a diluted serum sample to form a immune complex.
  • the immune complex is detected by a change in fluorescence polarization.
  • the assay involves only a single reagent, and a single step reaction in which an immune complex is detectable by a simple fluorescence polarization measurement.
  • the antigen reagent is also very stable with virtually indefinite lifetime when stored at refrigerated temperatures in the presence of a microbial inhibitor such as sodium azide.
  • the present method of the invention detects antibodies to bacterial antigens present in fluid comprising combining fluid which may contain antibodies to a bacterial antigen with a fluorophore-conjugated oligosaccharide antigen derived from the lipopolysaccharide antigen derived from the lipopolysaccharide fraction of a bacterial cell wall to form a mixture, incubating the mixture for a time sufficient to form an immune complex, and measuring the extent of formation of the immune complex by comparing the fluorescence polarization after complex formation to an unreacted control value.
  • the invention provides fluorophore-conjugated antigens comprising oligosaccharides derived from the lipopolysaccharide fraction of a bacterial cell wall.
  • the fluorophore-conjugated oligosaccharide is one embodiment derived from the O-antigen chain of the cell wall lipopolysaccharide fraction from bacteria, such as Gram negative bacteria, having us O-antigen chain.
  • the invention similarly comprises a homogeneous immunoassay in which antibodies to an antigen are reacted with an antigen to form an immune complex, the improvement to previous technology being a fluorophore-conjugated oligosaccharide antigen of bacterial cell wall forming a fluorescence polarization detectable immune complex with antibodies specific thereto.
  • the fluorophore-conjugated oligosaccharide antigens may be derived from any bacterium having a lipopolysaccharide cell wall fraction, as antigens derived from Gram negative bacteria such as B. abortus , Leptospira spp. , or Salmonella spp.
  • the fluorophore will have an emission life of 1 to 10 nanoseconds, and its emission may vary over a wave length of 400 to 800 nanometers. In the present invention, the fluorophore fluorescein is efficacious.
  • the assay of the present invention takes advantage of the well-known highly specific binding of antibodies to their corresponding antigens.
  • a bacterium invades an immunocompetent animal, certain structural/chemical moieties present on its surface are recognized as foreign by the immune system. Secretion of antigen-specific antibody ensues.
  • the population of antibodies is generally very heterogenous, with individual antibody molecules recognizing and binding different epitopes.
  • it is therefore important to select antigenic structures which contain the immunodominant epitopes, so that there is a strong likelihood of correctly identifying the largest possible number of positive sera.
  • Gram negative bacteria have cell walls that characteristically contain a diverse family of specialized glycolipids in which the saccharide forms repeating O-antigen chains, attached through non-repeating core glycan to lipid A, which is integrated into the outer membrane.
  • lipid A which is integrated into the outer membrane.
  • synthesis of the entire core sequence occurs by glycosyl transfers from sugar nucleotides, with addition at the nonreducing terminal and catalysis by glycosyl transferases.
  • the O-antigens generally act as surface antigens, and contain many of the immunodominant epitopes. These antigens also, when shed, behave as bacterial toxins.
  • the O-antigen chains are synthesized separately as modular glycans. The modules are spliced together before becoming linked to the core sequence.
  • the O-antigen chains are simpler than in Salmonella.
  • the O-antigen polysaccharides In one species of great interest from a diagnostics standpoint, B. abortus, the O-antigen polysaccharides have 1-2 linked 4,5-dideoxy-4-formamido-alpha-D-mannopyranosyl repeating units. The O-polysaccharide is extractible as an approximately 15,000-30,000 molecular weight molecule.
  • extraction methods for obtaining purified O-chain antigen reported in the literature for Gram negative bacteria generally. For Brucella abortus or Yersinia enterocolitica 0:9, phenol-water extraction, followed by dialysis and centrifugation may be used, as described in U.S. Patent No. 4,831,126 or 5,006,463.
  • the lipid fraction may be removed by extraction with acetone, ether-methanol, and chloroform successively, and then releasing the antigen fraction by alkaline hydrolysis. Further purification may be effected through ion exchange chromatography and gel filtration.
  • the length of polymer estimated to fill an antibody combining site is on the order of 4-6 sugar residues.
  • the O-antigen may be conveniently reduced in size by several methods, including acid or base hydrolysis, enzymes, oxidative or reductive processes, or by techniques known to result in selective cleavage at specific glycosidic linkages.
  • the general protocol for preparation of bacteria-derived polysaccharide antigen fragments useful in the present invention is:
  • DTAF was used to successfully label the 6-position of the sugar moiety in muramyl depeptide when all efforts to employ FITC had failed [Hiebert, C.K., et al., J. Med. Chem. 26:1729 (1983)].
  • Reductive amination of the terminal reducing sugar residue has been used to label carbohydrates with a variety of fluorophores including pyridylamine [Hase, S., et al., J. Biochem. 95:197 (1984)] and fluoresceinyl-ethylene diamine [Baynes, J.W., et al., Anal. Biochem. 170:382 (1988)].
  • fluorophores including pyridylamine [Hase, S., et al., J. Biochem. 95:197 (1984)] and fluoresceinyl-ethylene diamine [Baynes, J.W., et al., Anal. Biochem. 170:382 (1988)].
  • the carbohydrate and fluorophore, possessing an amino group are mixed together and the Schiff base formed is reduced to a stable amino linkage with sodium cyanoborohyride.
  • terminal aldehyde may be converted to an amino group by incubation with ammonium bicarbonate and then reacted with fluorophores such as carboxyfluorescein or dansyl chloride [Manger, I.D., et al., Biochemistry 31:10724 (1992)]
  • fluorophores such as carboxyfluorescein or dansyl chloride
  • Aldehydes may be generated in carbohydrates by periodate oxidation of vicinal hydroxyl groups [Rothfus, J.A. and Smith, E.L., J. Biol. Chem. 238:1402 (1963)].
  • RNA was labeled at its 3'-terminal ribose in this way with 5[[[2(carbohydrazino)methyl]thio]acetyl]-aminofluorescein [Janiak, F., et al., Biochemistry 31:5830 (1992)] and with rhodamine and fluorescein thiosemicarbazide [Bauman, J.G., J. Histochem. Cytochem. 29:227 (1981)].
  • the carbohydrate moiety of human alpha thrombin was labeled with aminoethyl and aminohexyl thioureal fluorescein [Carney, D.H., J. Supramol. Struc. 13:467 (1980)] and the cercarial glycocalyx of Schistosoma mansoni by fluorescein semicarbazide [Samuelson, J.C. and Caulfield, J.P., J. Cell Biol. 100:1423 (1985)].
  • Aldehydes may be generated and labeled at specific residues by combining enzymatic and chemical reactions.
  • galactose oxidase may be used to form the C-6 aldehyde on terminal D-galactose or N-acetyl-D-galactosamine residues [Avigad, E., et al., J. Biol. Chem. 237:2736 (1962)].
  • the same enzyme, in combination with neuraminidase was used to modify galactosyl residues penultimate to sialic acids and subsequently reacted with fluorescein ⁇ -alanine hydrazide [Wilchek, M. and Bayer, E.A., Methods Enzymol. 138:429 (1987)].
  • fluorophore is to a large extent a matter of convenience once a coupling chemistry has been selected. Virtually any fluorophore having a fluorescence life of between 1 and 10 nanoseconds, preferably 4 to 7 nanoseconds, and having a wavelength of 400 to 800 nanometers will have efficacy in the present invention.
  • the choice of fluorophores will influence the configuration and requirements of the optics of the instrument used to read the assays. It will also impact the optics technology utilized. For a detailed listing and discussion of a variety of interesting, commercially available fluorophores, see Handbook of Fluorescent Probes and Research Chemicals , ed. Karen Larison, by Richard P.
  • fluorophores in the labeled antigens of the present invention are fluorescein, rhodamine, BODIPYTM, Texas RedTM and Lucifer yellow. Fluorescein is most preferred for its convenience in screening O-antigen fragments for good assay candidates.
  • the fluorophore-conjugated oligosaccharide antigens are mixed with a serum sample diluted in a conventionally buffered solution, allowed to react for 1 to 3 minutes, and read in a fluorescence polarization instrument. Typically a background value is obtained prior to addition of the antigen reagent.
  • the assay is homogeneous, in that no further steps are required, as in heterogeneous assays in which antibody contained in the sample must be bound to antigen coated onto a solid surface, with subsequent washing and detection steps.
  • the assay is carried out at temperatures of 15°C to 37°C, but the assay may be carried out under any temperature and reaction conditions which permit specific immune complex formation.
  • fluorescence polarization a molecule conjugated to a fluorophore is present in solution.
  • the molecular rotation of the molecule in solution is a function of its size and the viscosity of the solution. If the fluorophore-conjugated molecule is relatively small compared to its size when bound to a larger molecule, such as an antibody, the fluorescence emitted by such small molecule will be relatively less polarized than when it is bound. This is because the complexed ligand-target molecule is relatively large, and rotates more slowly. Hence, more of the emitted light is directed in one geometric plane, and the fluorescence polarization value increases.
  • FP has the following important characteristics: it detects change in size of molecules, it detects change in viscosity of the medium, it may be used to detect and measure binding of small molecules to big molecules. In addition, it has broad application in detection and measurement of the conversion of large molecules to small molecules.
  • the FP assay is homogeneous, (reactions take seconds to minutes), utilizes stable reagents, (no enzymes, substrates or radioisotopes) and is highly automatable, (single incubation, a single reagent is possible, and no washing steps are required).
  • the column was eluted with 0.01 M phosphate buffer, pH 7.4. Two fractions were collected. The first was buffer (7 mL) and the second, a bright green fluorescent fraction (tracer I10-1; 7 mL). The elution buffer was then changed to 0.1 M phosphate, pH 7.4. Two fractions were collected. The first was buffer (10 mL) and the second, a bright green fluorescent fraction (tracer I10-2; 25 mL). The latter fraction contained the best tracer. Tracer ⁇ l/assay mP -ve mP Strong +ve mP Weak +ve 16469 I10-1 0.5 84.8 176.4 110.4 I10-2 1.5 80.9 225.7 138.6 Therefore, the yield is approximately 5000 assays per mg.
  • Monoclonal antibodies cannot be used to screen tracers for reactivity to antibodies generated by natural infection in bovines, as shown in Table 2.
  • Tracer 94082502 reacts strongly with mAB ST9, specific for the O-polysaccharide from B. abortus .
  • POS1, POS3 reacts strongly with a strong positive (POS4).
  • POS4 reacted strongly with all of these samples, but poorly with mAb YST9.
  • a fluorescence polarization assay for the detection of antibodies to M. paratuberculosis and M. bovis was compared to a reference ELISA (ADRI). Results compared favorably with some exceptions as seen in Table 3.
  • Negative samples 54 was positive in ELISA (high cutoff) but negative by FP.
  • Positive samples 52C8, 27A9 and 194F9 were negative by ELISA (high cutoff) and positive by FP (105 mP cutoff).
  • Using the low cutoff ELISA was positive on positive samples 36-33 and FP was negative; samples 52C8 and 27A9 became positive.
  • negative samples 103E9, 1.06E9, 54 and 56 became positive.
  • Assay sensitivity and specificity for the FPIA before and after decoding of the serum samples are as follows. Using a preliminary cutoff of 107.2 mP, the point estimates of sensitivity and the specificity of the FPA after initially testing all samples were 98.2+/-1.1% and 99.8+/-0.09%, respectively. After decoding and retesting false positive and false negative samples, the values increased to 98.5+/-1% and 100%, respectively.
  • Figures 1 and 2 represent histograms of the distribution of results obtained with the positive and negative sera in the FPA before and after decoding of the samples. While the cutoff from Figure 1 is difficult to determine because of the overlap between positive and negative samples in the 90 to 140 mP region, it is clear that the cutoff should be above 100 mP and a 107.2 value as calculated using the 100th percentile of the negative sera is not unreasonable. From Figure 2, however, after retesting 24 false positive samples, it is apparent that the cutoff should be set at a lower level. If a value of 90 mP is selected, three false negative results and one false positive are evident. These findings were confirmed using ROC analysis ( Figure 3). Thus, if a cutoff of 90 mP is selected, the sensitivity and specificity values are 99.02 and 99.96%, respectively.
  • the four control sera, a strong positive, a weak positive, a negative and a serum from a vaccinated animal, included in each 100 samples tested were plotted in Figures 4A, B, C and D. Each point represents an individual determination. The upper and lower lines for each set of points indicate +/- 2 standard deviations of the mean.
  • 2 gave positive reactions in the FPIA using cutoff of 90 mP, giving a point specificity value of 99.2%.
  • the specificity in the BPAT was 48.6%
  • the CFT was 49.0% (87 of 250 sera were anticomplementary and no diagnosis could be made)
  • the I-ELISA was 56.3%
  • the C-ELISA was 97.2%.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (7)

  1. Méthode pour détecter la présence d'anticorps contre un antigène bactérien dans un échantillon de fluide, comprenant les étapes consistant :
    à combiner un fluide qui peut contenir des anticorps contre un antigène bactérien avec un antigène oligosaccharidique conjugué à un fluorophore, ledit antigène oligosaccharidique d'un polysaccharide d'antigène O de la fraction lipopolysaccharidique d'une paroi de cellule bactérienne ayant une masse moléculaire allant de 1000 à 30 000 daltons pour former un mélange ; et
    à incuber ledit mélange pendant une durée suffisante pour former un complexe immun, et à mesurer l'ampleur de la formation dudit complexe immun en comparant la valeur de polarisation de fluorescence après formation du complexe à une valeur de témoin négatif.
  2. Antigènes bactériens conjugués à un fluorophore consistant en :
    un oligosaccharide, ledit antigène oligosaccharidique d'un polysaccharide d'antigène O de la fraction lipopolysaccharidique d'une paroi de cellule bactérienne ayant une masse moléculaire allant de 1000 à 30 000 daltons; et
    un fluorophore, lesdits antigènes conjugués à un fluorophore étant capables de se lier à des anticorps spécifiques desdits antigènes bactériens pour produire un changement détectable en polarisation de fluorescence.
  3. Antigènes bactériens conjugués à un fluorophore de la revendication 2, dans lesquels ledit fluorophore est choisi dans le groupe consistant en fluorescéine, rhodamine, BODIPY™, Texas Red™ et jaune Lucifer.
  4. Méthode de test immunologique homogène pour détecter la présence d'anticorps spécifiques d'antigènes bactériens dans un échantillon de fluide, dans laquelle lesdits anticorps spécifiques d'antigènes bactériens se lient à des antigènes marqués, caractérisée en ce que ladite méthode implique l'utilisation d'antigènes oligosaccharidiques conjugués à un fluorophore, ledit antigène oligosaccharidique d'un polysaccharide d'antigène O des lipopolysaccharides d'une paroi de cellule bactérienne ayant une masse moléculaire allant de 1000 à 30 000 daltons, dans laquelle lesdits antigènes oligosaccharidiques conjugués à un fluorophore se lient auxdits anticorps pour produire un changement détectable en polarisation de fluorescence.
  5. Méthode des revendications 1 ou 4, dans laquelle ledit antigène oligosaccharidique conjugué à un fluorophore est dérivé de la chaíne d'antigène O des lipopolysaccharides de bactéries gram-négatives.
  6. Méthode des revendications 1 ou 4, dans laquelle ledit antigène oligosaccharidique conjugué à un fluorophore est dérivé de la chaíne d'antigène O des lipopolysaccharides de Brucella abortus.
  7. Méthode des revendications 1 ou 4, dans laquelle ledit antigène oligosaccharidique conjugué à un fluorophore est dérivé des antigènes arabinomannane sans lipides de Mycobacterium spp.
EP96933797A 1995-08-28 1996-08-28 Detection d'anticorps contre des antigenes bacteriens par polarisation de fluorescence Expired - Lifetime EP0788605B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US520313 1995-08-28
US08/520,313 US5976820A (en) 1995-08-28 1995-08-28 Detection of antibodies to bacterial antigens by flourescence polarization
PCT/US1996/014860 WO1997008559A1 (fr) 1995-08-28 1996-08-28 Detection d'anticorps contre des antigenes bacteriens par polarisation de fluorescence

Publications (2)

Publication Number Publication Date
EP0788605A1 EP0788605A1 (fr) 1997-08-13
EP0788605B1 true EP0788605B1 (fr) 2004-02-04

Family

ID=24072066

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96933797A Expired - Lifetime EP0788605B1 (fr) 1995-08-28 1996-08-28 Detection d'anticorps contre des antigenes bacteriens par polarisation de fluorescence

Country Status (6)

Country Link
US (2) US5976820A (fr)
EP (1) EP0788605B1 (fr)
DE (1) DE69631468T2 (fr)
ES (1) ES2215197T3 (fr)
PT (1) PT788605E (fr)
WO (1) WO1997008559A1 (fr)

Families Citing this family (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5976820A (en) 1995-08-28 1999-11-02 Jolley; Michael E. Detection of antibodies to bacterial antigens by flourescence polarization
DE19842609C2 (de) * 1998-09-17 2000-09-21 Diagnostik Gmbh Leipzig Lab Salmonellenantigengemisch und Kit zur Bestimmung von Antikörpern gegen Salmonellen
US6350574B1 (en) 1998-09-23 2002-02-26 Ronald C. Montelaro Fluorescence polarization—based diagnostic assay for equine infectious anemia virus
DE10023051B4 (de) * 2000-05-11 2004-02-19 Roche Diagnostics Gmbh Verfahren zur Herstellung von Fluoresceinisothiocyanat-Sinistrin, dessen Verwendung und Fluoresceinisothiocyanat-Sinistrin enthaltende diagnostische Zubereitung
EP1881324B1 (fr) 2000-09-11 2015-01-21 Diachemix LLC Analyse homogène à base de polarisation fluorescente pour détermination désoxynivalénol en grains
US6482601B1 (en) * 2000-09-11 2002-11-19 Diachemix Llc Fluorescence polarization-based homogeneous assay for fumonisin determination in grains
US6812036B2 (en) 2000-09-11 2004-11-02 Diachemix Llc Fluorescence polarization-based homogeneous assay for deoxynivalenol determination in grains
NO20006130D0 (no) * 2000-12-01 2000-12-01 Erling Sundrehagen Reagens og analysemetode
ATE373240T1 (de) * 2001-07-13 2007-09-15 Diachemix Llc Auf fluoreszenzpolarisation beruhender homogener assay für aflatoxine
AU2002336705B2 (en) * 2001-10-31 2006-06-15 Diachemix Llc A rapid serological test for paratuberculosis using fluorescence polarization technology
EP1461078B1 (fr) * 2001-10-31 2011-12-21 Diachemix LLC ANALYSE DE POLARISATION DE FLUORESCENCE A BASE DE PEPTIDES DESTINEE A LA DETECTION D ANTICORPS CONTRE i MYCOBACTERIUM BOVIS /i
RU2224257C1 (ru) * 2002-07-01 2004-02-20 Федеральное государственное унитарное предприятие "Пермское научно-производственное объединение "Биомед" Способ диагностики сальмонеллеза
US20040137525A1 (en) * 2002-10-16 2004-07-15 Jolley Michael E. Detection of salmonella cells by fluorescence polarization
AU2003287153A1 (en) * 2003-11-05 2005-06-24 The United States Of America As Represented By The Secretary Of The Navy Naval Medical Research Center Fluorescence polarization instruments and methods for detection of exposure to biological materials by fluorescence polarization immunoassay of saliva, oral or bodily fluids
US7408640B2 (en) * 2003-11-05 2008-08-05 The United States Of America As Represented By The Secretary Of The Navy Fluorescence polarization instruments and methods for detection of exposure to biological materials by fluorescence polarization immunoassay of saliva, oral or bodily fluids
DE502005005099D1 (de) * 2005-03-23 2008-10-02 Roche Diagnostics Gmbh Verfahren zur Bestimmung der Glucosekonzentration durch Fluoreszenzpolarisation
US9006283B2 (en) * 2007-07-12 2015-04-14 Acumen Pharmaceuticals, Inc. Methods of modifying amyloid β oligomers using non-peptidic compounds
US8962677B2 (en) * 2007-07-12 2015-02-24 Acumen Pharmaceuticals, Inc. Methods of restoring cognitive ability using non-peptidic compounds
US20110098309A1 (en) * 2007-07-12 2011-04-28 Acumen Pharmaceuticals, Inc. Methods of inhibiting the formation of amyloid-beta diffusable ligands using acylhydrazide compounds
DE102007038773A1 (de) 2007-08-16 2009-03-12 Zf Friedrichshafen Ag Verfahren zur Durchführung einer zugkraftunterbrochenen Schaltung bei einem parallelen Hybridfahrzeug
DE102007041569A1 (de) 2007-09-01 2009-03-05 Zf Friedrichshafen Ag Verfahren zum Steuern und/oder Regeln einer Hybridantriebsanordnung
WO2009079566A2 (fr) 2007-12-18 2009-06-25 Acumen Pharmaceuticals, Inc. Nouveaux polypeptides du récepteur de l'addl, polynucléotides et cellules hôtes pour une production recombinante
US7850917B2 (en) 2008-03-11 2010-12-14 Ortho-Clinical Diagnostics, Inc. Particle agglutination in a tip
US20090270269A1 (en) * 2008-04-28 2009-10-29 Ashok Kumar Nano-scale fluoro-biosensors exhibiting a low false alarm rate for rapid detection of biological contaminants
US20100015690A1 (en) 2008-07-16 2010-01-21 Ortho-Clinical Diagnostics, Inc. Use of fluid aspiration/dispensing tip as a microcentrifuge tube
EP2663338B1 (fr) 2011-01-14 2024-03-06 Emory University Oligosaccharides à base de glucose comprenant un radionucléide émettant des positrons et leur utilisation pour l'imagerie
CN102662049B (zh) * 2012-05-07 2014-03-26 清华大学 基于碳纳米材料的免疫分析方法
US9402925B2 (en) 2013-03-12 2016-08-02 The Board Of Trustees Of The Leland Stanford Junio Probes and methods of imaging a bacterial infection
GB201306882D0 (en) 2013-04-16 2013-05-29 Sec Dep For Environment Food & Rural Affairs Acting Through The Oligosaccharide
US10048256B2 (en) 2015-03-18 2018-08-14 Bio-Rad Laboratories, Inc. Sample analysis systems and methods
EA201990945A1 (ru) 2016-11-04 2019-09-30 Зе Секретари Оф Стейт Фор Энвайронмент, Фуд Энд Рурал Эффейрс Олигосахариды с колпачковой структурой, содержащие семь или более звеньев из 4,6-дидезокси-4-ациламидо-αльфа-пиранозы, и их конъюгаты в качестве вакцин, направленных против инфекций, вызванных организмами brucella
CN106353496A (zh) * 2016-11-10 2017-01-25 中国兽医药品监察所 布鲁氏菌荧光偏振（fpa）检测试剂盒

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1212051A (fr) * 1984-03-02 1986-09-30 John W. Cherwonogrodzky Identification de brucella abortus et de la brucellose
CA1336576C (fr) * 1986-09-26 1995-08-08 Malcolm B. Perry Immunodosages pour distinguer les animaux atteints de la brucellose et ceux qui sont vaccines
GB8624253D0 (en) * 1986-10-09 1986-11-12 Harvey W Endotoxin assay
US4921878A (en) * 1987-06-05 1990-05-01 Pall Corporation Non-fluorescing, non-reflective polyamide for use in diagnostic testing
US5212298A (en) * 1989-08-16 1993-05-18 Monsanto Company Method for producing synthetic N-linked glycoconjugates
US5445935A (en) * 1992-11-23 1995-08-29 Royer; Catherine A. Quantitative detection of macromolecules with fluorescent oligonucleotides
DE4240056A1 (de) * 1992-11-28 1994-06-01 Boehringer Mannheim Gmbh Streptolysin O Peptidantigene und Verfahren zur Bestimmung von Streptolysin-Antikörper
US5610075A (en) * 1995-01-17 1997-03-11 Stahl-Rees; Marianne Competitive electrochemiluminescence assays for endotoxins using a ruthenium label
US5976820A (en) 1995-08-28 1999-11-02 Jolley; Michael E. Detection of antibodies to bacterial antigens by flourescence polarization

Also Published As

Publication number Publication date
DE69631468D1 (de) 2004-03-11
DE69631468T2 (de) 2004-10-07
PT788605E (pt) 2004-06-30
EP0788605A1 (fr) 1997-08-13
WO1997008559A1 (fr) 1997-03-06
US5976820A (en) 1999-11-02
ES2215197T3 (es) 2004-10-01
US6596546B1 (en) 2003-07-22

Similar Documents

Publication Publication Date Title
EP0788605B1 (fr) Detection d'anticorps contre des antigenes bacteriens par polarisation de fluorescence
JP2667695B2 (ja) 試料中のエンドトキシンの存在を決定する方法
US4652518A (en) Diagnosing chlamydia infections with Re-lipopolysaccharide complexed to carrier or antibody thereto
US4882423A (en) Substance-conjugated complement component C1q
JPH0731199B2 (ja) 低 ｐＩ蛋白質または炭水化物を含んでなる特異的バインディング組成物ならびに診断試験キットおよび使用方法
Carlsson et al. Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA)
IL186722A (en) Method for immobilization of a polysaccharide on a detection system carrier, a detection system carrier comprising an immobilized polysaccharide-protein complex and a method for determining the presence of a gram negative bacterium in a sample
Lilenbaum et al. Evaluation of an ELISA-PPD for the diagnosis of bovine tuberculosis in field trials in Brazil
US4711841A (en) Method for determining one or more antigens in a sample
DK174032B1 (da) Sæt samt fremgangsmåde til immunometrisk dosering, der kan anvendes på hele celler
US4831126A (en) Antigenic polysaccharide specific to Brucella abortus and Yersinia enterocolitica serotype 0:9
CN101592660B (zh) 布鲁氏菌病间接酶联免疫吸附试验奶液抗体检测试剂盒
JPS6097272A (ja) 免疫螢光法
EP2261666B1 (fr) Procédé de détection de pneumocoque
US7713715B2 (en) Method for diagnosing infections
CN101799470A (zh) 布鲁氏菌病间接酶联免疫吸附试验抗体检测试剂盒
BR112012030535B1 (pt) Método para detectar um componente da parede celular de levedura
EP2084261A1 (fr) Analyse pour les anticorps envers le bacille de koch
CA1083480A (fr) Identification de neisseria gonorrhoeae a l'aide d'anticorps contre un antigene lipopolysaccharidique
CA1336576C (fr) Immunodosages pour distinguer les animaux atteints de la brucellose et ceux qui sont vaccines
Svenungsson et al. Diagnosis of Salmonella infections: specificity of indirect immunofluorescence for rapid identification of Salmonella enteritidis and usefulness of enzyme-linked immunosorbent assay
JPS60259966A (ja) 細菌および菌類感染の迅速検出方法
EP0134868A1 (fr) Organismes unicellulaires activés par glutaraldéhyde comme supports solides pour agents sensibilisateurs et usages d'organismes unicellulaires sensibilisés
AU2012297533A1 (en) Method of diagnosing bacterial infections using bacterial glycoproteins
RU2137133C1 (ru) Экспресс-способ оценки функциональной активности b-системы иммунитета по гевондян

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19970528

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): DE ES FR GR IT NL PT

17Q First examination report despatched

Effective date: 20000215

RTI1 Title (correction)

Free format text: DETECTION OF ANTIBODIES TO BACTERIAL ANTIGENS BY FLUORESCENCE POLARIZATION

RTI1 Title (correction)

Free format text: DETECTION OF ANTIBODIES TO BACTERIAL ANTIGENS BY FLUORESCENCE POLARIZATION

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

RTI1 Title (correction)

Free format text: DETECTION OF ANTIBODIES TO BACTERIAL ANTIGENS BY FLUORESCENCE POLARIZATION

RTI1 Title (correction)

Free format text: DETECTION OF ANTIBODIES TO BACTERIAL ANTIGENS BY FLUORESCENCE POLARIZATION

RTI1 Title (correction)

Free format text: DETECTION OF ANTIBODIES TO BACTERIAL ANTIGENS BY FLUORESCENCE POLARIZATION

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: DIACHEMIX LLC

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): DE ES FR GR IT NL PT

REF Corresponds to:

Ref document number: 69631468

Country of ref document: DE

Date of ref document: 20040311

Kind code of ref document: P

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20040401402

Country of ref document: GR

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20040429

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2215197

Country of ref document: ES

Kind code of ref document: T3

ET Fr: translation filed
PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20041105

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES;WARNING: LAPSES OF ITALIAN PATENTS WITH EFFECTIVE DATE BEFORE 2007 MAY HAVE OCCURRED AT ANY TIME BEFORE 2007. THE CORRECT EFFECTIVE DATE MAY BE DIFFERENT FROM THE ONE RECORDED.

Effective date: 20050828

PGRI Patent reinstated in contracting state [announced from national office to epo]

Ref country code: IT

Effective date: 20091201

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GR

Payment date: 20140827

Year of fee payment: 19

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20150826

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20150827

Year of fee payment: 20

Ref country code: ES

Payment date: 20150826

Year of fee payment: 20

Ref country code: PT

Payment date: 20150804

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20150817

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20150825

Year of fee payment: 20

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160303

REG Reference to a national code

Ref country code: GR

Ref legal event code: ML

Ref document number: 20040401402

Country of ref document: GR

Effective date: 20160303

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 69631468

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: MK

Effective date: 20160827

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20160906

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20161205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20160829